POS0333 NLRP12 EXACERBATES OSTEOARTHRITIS BY PROMOTING DEGRADATION OF NOD2 IN SYNOVIAL MACROPHAGES
Authors: C. LI, S. Lin, Z. Ouyang, T. Liu, Z. Zheng, J. Xu, Y. Ding
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Osteoarthritis is the most common degenerative bone and joint disease. Hypothesis has been widely accepted that osteoarthritis (OA) starts from cartilage injury and loss, while emerging evidences suggest that synovial inflammation precedes cartilage loss during the progression of OA [1]. Activation of macrophages plays a crucial part in synovial inflammation in OA [2]. In our previous researches, we have demonstrated NOD2 as an inhibitor of macrophage activation <i>in vitro</i>. Besides, bioinformatic analysis suggested a potential link between NLRP12 and NOD2. <h3>Objectives</h3> To investigate the role of NOD2 in OA <i>in vivo</i> and explore the potential interaction between NOD2 and NLRP12. <h3>Methods</h3> We established CIOA (Collagenase-induced OA) model with 8-week-old C57BL/6J mice, and injected NOD2 over-expression (oe-NOD2) lentiviral vectors into the knee joint cavity as experimental group (CIOA + oe-NOD2), with the empty vectors as control (CIOA + Mock). 8 weeks later, the knee joints were harvested and stained with Safranin O/fast green. Besides, three-dimensional reconstruction of Micro-CT images were employed to evaluate the pathological changes of OA. In addition, we applied lentiviral transfection, co-immunoprecipitation (Co-IP), and ubiquitination assays to investigate the interaction between NOD2 and NLRP12, as well as its mechanism of action in the regulation of macrophage activation. <h3>Results</h3> <i>In vivo</i> over-expression of NOD2 showed significant inhibition of pathological changes (Figure 1A, 1B). Though NLRP12 had no impact on mRNA level of NOD2 in RAW264.7 macrophages (Figure 1C), NOD2 expression at protein level was negatively correlated with NLRP12 (Figure 1D), suggesting that NLRP12 may influence the degradation of NOD2. Co-IP experiments also confirmed the existence of interaction between NLRP12 and NOD2 at protein level, which was influence by MG132, inhibitor of ubiquitin-proteasome pathway (Figure 1E). Besides, NLRP12 over-expression impaired inhibition of macrophage inflammation by NOD2 (Figure 1F). Since HSP90 binds NOD2 to prevent its proteasomal degradation via poly-ubiquitination (Poly-Ub) [3], we therefore performed Co-IP and Poly-Ub assays. NLRP12 was associated with reduced binding of HSP90 with NOD2, greater scale of K48 Poly-Ub assembling on NOD2, and thus lower level of NOD2 protein. Furthermore, proteasomal degradation of NOD2 was blocked by MG132, though interrupted binding between NOD2 and HSP90, and accumulation of Poly-Ub chains (Figure 1G). These findings suggested sequestration of HSP90 from binding NOD2 by NOD2-NLRP12 interaction, which leaded to assembly of K48 Poly-Ub chains onto NOD2, and its proteasomal degradation (Figure 1H). <h3>Conclusion</h3> NOD2 was demonstrated as an inhibitor of OA <i>in vivo</i>, in consistence with our previous <i>in vitro</i> data. Besides, NLRP12 interacted with NOD2, and promoted its Poly-Ub and subsequent proteasomal degradation. Our findings highlighted the impressive potential of NLRP12 to be a preventative and therapeutic target in OA, though more in-depth investigations are indispensable before further conclusion is reached. <h3>References</h3> [1]Atukorala I, <i>et al</i>. Ann Rheum Dis. 2016;75(2):390-5 [2]Sanchez-Lopez E, <i>et al</i>. Nat Rev Rheumatol. 2022,18(5):258-75 [3]Lee KH, <i>et al</i>. J Biol Chem. 2012;287(47):39800-11 <h3>Acknowledgements</h3> I would like to thanks all people who have helped and were directly or indirectly involved in this study. This work was supported by Guangdong Medical Research Foundation [A2020094], Guangdong Basic and Applied Basic Research Foundation [2021A1515110996], and Science and Technology Project of Guangzhou [202102020132, 202206010140]. <h3>Disclosure of Interests</h3> None Declared.